Preparation of highly potent vitamin concentrates



vitamins only with Patented A r; 24, 1945 UNl-TED STAT as PATENT OFFICE PREPARATION OF HIGHLY POTEN T VITAMIN CONCENTRA'IES lLoran 0. Buxton,

Belleville, N. .L, assignor to National Oil Products Company, Harrison, N. .L, a corporation of New Jersey No Drawing. Application September 12, 1 941,

v ,Serial No. 410,579

11 Claims. (01. zen-397.2)

This invention relates in general to the preparation of vitamin concentrates, and more partic-- ularly to the preparation of highly-potent concentrates of vitamin A andhighly potent concentrates of vitamin D from'fish liver and other marine oils. I

o It is well known that the fish liver and other marine oils constitute the richest natural sources of vitamins A and D. For some time it has been a common practice to saponify these oils and to characteristics and molecucan be separated from the extreme dimculty. At the is no practical commercial a satisfactory separation of present time theremethod for e ecting action of asuitable acylating agent in such a manner that only the vitamin A alcohols present are esterified to the substantial exclusion of the vitamin D alcohols present. This esterification reaction may be controlled by supplying only the stoichiometrical quantity of the acylating agent 'to accomplish the foregoing results, or if an excess quantity of the acylating agent is used, the

reaction is arrested at the proper point to prevent any substantial esterification oi the vitamin D. Vitamin A, which is a primary alcohol, is more reactive towards esteriflcation than the vitamin D which is a secondary alcoholyconsequently so similar to vitamins A and these constituents from the vitamins. Moreover,

there has hitherto been no commercially practical method for separating the vitamin A from thevitamin D. -It has been proposed to separate these two vitamins from each other by a large number of successive fractional crystallizations. However, such a method, although it will give fairly satisfactory results on a laboratory scale, is not feasible-for commercial practice as it is too costly, complicated and time-consuming.

It is the object of this invention to provide a relatively simple and efiicient process for preparing highly potent concentrates of vitamin A and highly potent concentrates of vitamin D from fish and fish livero A further object of this invention'is to provide a commercially the treatment of the unsaponifiable portion of fish liver and other marine oils to separate the vitamins contained therein from the non-vitamin materials.

Another object or this invention is to provide a commercially practicable, efilcient process for the treatment of vitamin A and D-containing materials 'to recover the vitamins A and D individually. i

Other objects of theinvention will in part be obvious and will in part appear hereinafter.

The foregoing and other objects of the invention may be realized by subjecting the unsaponiflable fraction of a practicable, efiiclent process for i fish'on fish liver oil to the u been removed and selective esterification can be effected as outlined above. Upon completion of the esteriilcation procedure the mass is dissolved in a hydrocarbon solvent and this solution then extracted with a solvent which is characterized by being miscible with the esterifled constituents and immiscible with the hydrocanbon solvent and the unesterifled components. After the vitamin A esters are removed, the residual hydrocarbon solution is again extracted with a second solvent which is immiscible with the hydrocarbon solvent and inactive hydrocarbon materials, such as squalene,

which are present in the unsaponifiable material, but which will preferentially extract the vitamin D. The solvent is removed from the vitamin A esters and the residue saponifled, and the vitamin A alcohols removed from the saponified mass by extraction with a suitable solvent. If desired, the saponification maybe carried out before removing the solvent as in some cases the vitamin A may be more readily extracted if this is done. The reason for saponifying the vitamin A esters and thus converting them to their alcoholic form is that the vitamin esters produced during the esterification step are biologically inactive. The solvent is also removed from the solution containing the vitamin D and the residue is treated in the same -manner as was the residue containing the vitamin A esters in order to reactivate any vitamin D that may have been the process is properly carried out, very little or none of the vitamin D will be esterified, and, if desired, the saponification of the vitamin D residue may well be omitted. In either case a very highly potent vitamin D concentrate is obtained,

which contains a relatively small quantity, if any, of vitamin A. The vitamin A concentrate will contain little or no vitamin D and only a very small amount of inactive material. The above procedure may be modified slightly by esterifyingthe vitamin D after the vitamin A esters have before the vitamin D is recontaining the vitamin A esters 1 esterified. However, if

, step and remove moved from the hydrocarbons such as squalene. The vitamin D esters may then be saponified as in the caseof thevitamin A esters.

Y The invention accordingly comprises the several Steps and the relation of one or more of such steps with respect to each of the others thereof, which will be exemplified in the process hereinafter disclosed, and the scope of the invention will be indicated in the claims.

In carrying out the process ofthe invention, it is preferred to start with an unsaponiflable fraction which is substantially devoid of cholesterol and other relatively high melting sterols. The procedure for removingcholesterol is well known and is usually effected by dissolving the vitamin fraction in a solvent and lowering the tempera- I ture of the solution to freeze out the cholesterol.

The remova1 of cholesterol, the esteriflc-ation of the vitamin A, and the extraction of the vitamin A esters and vitamin D may all be carried out in the same solvent vehicle- A few' of the solvents which may be so used are the aliphatic and allcyclic hydrocarbons, including, among others, pentane, hexane, heptane, octane, nonane, etc., petroleum ether, cyclohexane, methyl cyclohexane, etc., as well as compatible mixtures of these and/or other suitable solvents.

Usually it is preferred to use several difierent solvents in carrying out the complete process; thus in removing the cholesterol, it is preferred, to dissolve the unsaponifiable material in methanol as it is one of the most eflicient solvents for that purpose. At temperaturessubstantially below room temperature, 1. e. C. or lower, cholesterol is quite insoluble in methanol and precipitates out from the solution, whereas vitamin A and vitamin D are quite soluble. Since methanol is not a suitable medium in which to carry out the esterification step, it is removed in a suitable way, e. g. vacuum distillation, and the residue redissolved in another solvent which will be a suitable medium for the esteriflcation procedure. Other solvents which may well bevent, such as acidified 50% methanol, after which the selective extraction of the vitamin A esters and vitamin D is carried out. Solvents suitable as extractionmedia include, inter alia, heptane, octane, nonane, hexane, pentane, petroleum ether, cyclohexane, methyl cyclohexane, etc.

Various acylating agents may be used in carry- I is not limited to these esterifying agents as any obtain the desired results 7 conditions.

usedin place of methanol are ethanol, isopropajnol, methyl acetate, ethyl acetate, methyl formate, ethyl formate, etc. Solvent vehicles which are suitable for the .esteriflcation step include, inter alia, pyridine, benzene, tane, hexane, pentane, octane, nonane, cyclohexan'e, methy1 cyclohexane, etc., and compatible mixtures of these and/or other suitable solvents. Preferably, an esterifleation catalyst is added to the solution in order to accelerate this reaction. If pyridine is used as the solvent vehicle it is not necessary to add an esteriiication catalyst as pyridine itself functions as a catalyst. In fact in some cases where a solvent other than pyridine is used, pyridine is preferably added as the esterification catalyst. Other compounds inaddition to or in lieu of pyridine maybe used as esterification catalysts, such as quinoline, piperidine, etc. Usually if either pyridine or benzene is used as the esteriflcation medium, it is removed from the reaction mixture and the mixture dissolved in another solvent before proceeding with the extraction step. If benzene has been used it may easily be removed by a convenient method such as vacuum distillation and .the residue then dissolved in the solvent in carry out the extraction tep. In the .case of pyridine, quinoline, piperidine, etc. it is preferable petroleum ether, hepwhich it is desired to it is preferred not 'to us or compatible mixture of such agents which will produce vitamin A esters which may be selectively extracted out from a hydrocarbon solvent solutlon containing vitamin D alcohols and hydrocarbons such. as squalene may be used.

It is preferred to use amount of acylating a ent somewhat in excess of the stoichiometrical quantity necessary to est'erify all of the vitamin A, and then to vitamin A has been esterinedand before any'or only very little of the vitamin-D has been esterified. However, if desired. only the stoichiometrical amount of the'acylating agent necessary to esterify the vitamin A may be added, and the reaction allowed to proceed to equilibrium. It is somewhat more diflicult to determine the exact amount of acylating -agent to use in order to acylating agent to arrest the reaction when an excess just when esterifying agent is used under uniform amount 01 The esterincation process may very well be carried out at room temperature and if it is desired to accelerate this reaction, it may be carried out at slightly elevated temperatures. It is desirable to use uniform conditions of temperature, reagents, etc. as it must be determined exper mentally just when'to arrest the reaction. Naturally, therefore, the best results will be obtained 3 when the process is consistently carried out in a uniform manner.

When the esterification reaction has reached the proper point it may be arrested in any suitable manner. If'an esterification catalyst has been used, it may be readily removedby washin the reaction mixture with an acidified methanol solution. It is preferred to methanol solution containing about 5% acetic acid. Besides thus removing the esterification catalyst, a large part of the acylating agent will also be removed and the esterification reaction for all practical purposes will be concluded. As benzene or pyridine as solvents in which to carry out the extraction step. if they have been used as the esterifying medium they may be removed as described he'reinabove.

and the residue redissolved in another solvent as previously described. I 1

The extraction of the partially esterifled mass is preferably carried out in an aliphatic or allcyclic hydrocarbon solvent vehicle such as heptane, octane, nonane, hexane, pentane, petroleum ether, cyclohexane, methyl cyclohexane, and the like. Also compatible mixtures of theseand/or other suitable solvents may be used. The exarrest the reaction when all of the than it is to determine I use about a 50% free esters.

panol, diacetone alcohol, other alcohols, and acetic acid, the percentage of water in each solvent being predetermined to yield the desired extractive powers with respect to the vit A and D components, respectively. In order to selectively extract first the vitamin A esters and then the vitamin D, varying strength solutions of the above solvents are used, c. g. to extract the vitamin A esters, about a 90% methanol solution may be used, and then to extract the vitamin D about a 95% methanol solution may be used. When acetic acid is used it has been found that solutions containing about 83% acetic acid will extract the vitamin A esters and 90% will extract the vitamin D. If desired, one solvent may be used for the first series of extractions and a different solvent used for the second series. The number of extractions to be carried out in each case will depend generally upon the type of solvents used, th amount of material to be extracted,.the temperature at which the extracting is done, the type of material being extracted, etc. Ether a continuous or a successiv extraction process may be used. In some cases the extraction medium and extracting agent may be slightly soluble in each other. Ifso, it is preferred that they b saturated with'each other before being used since better results will usually 'be obtained by so doing. Extracting agents other than those mentioned above may be used; the principal prerequisites being thatsuch an agent'be immiscible, or relatively so, with respect to' the extraction medium, and that it will selectively extract the constituent or constituents desired. I

The extraction steps may be carried out at room temperature; however, in some instances it may be preferred to extract at a temperature substantially below room temperature, since more efilcie'nt results will usually be obtained and fewer extractions will be required.

When a dicarboxylic anhydride is used as the ,acylating agent, it may be desirable to treat the reaction mixture with an alkali metal compound before the vitamin A esters are extracted therefrom. The reason that it may be desirable to do this is because in the process of esterification there may be produced on the anhydride, one free carboxyl group which is not esterified and by treating the reaction mixture with an alkali be neutralized and alkali metal salts of the vitamin A esters produced. Such salts are usually more soluble in the solvents which are used to extract the vitamin A esters than are the saltmethanol may be used to extract the alkali metal salts of the vitamin A esters, whereas to extract the esters as such, 90% methanol should be used. Thus a better separation of theesterifled material from the une'sterifled material may be obtained as the unesterified material is much less soluble in the less concentrated solvents. Naturally only enough of the'alkali metal compounds to neutralize the free carboxyl group should be used, as an excess will serve to split the esters.

As the vitamin A esters produced are biologically inactive, they must be saponified to convert the same to their alcohol form. The vitamins are then extracted from the saponified msss with a. suitable solvent, such as ethylene dichloride,

- petroleum ether, sulfuric ether, etc.

For a fuller understanding of the nature and objects of the invention, reference should be had Thus, for example, 80% to 85%" room temperature for two hours,

to the following examples which are given merely to further illustrate the invention and are not to be construed in a limiting sense, all parts given being by weight, 7

saponifiable fraction 01' cod liver oil previously freed of cholesterol and containing 130,000 U. S. P.

,units vitamin D/. and 380,000 U. S, P. units vitamin A/gm. were added. The mixture was left to stand in the dark with occasional shaking at 300 parts of petroleum ether were added and the pyridine removed by washing with 50% methanol contain ing 5% acetic acid. The petroleum ether fraction was thereafter washed twice with 70% methanol. The same was extracted with fifteen 200 part portions of 90% methanol. The 90% methanol extracts were-combined and the solvent removed, thus yielding a residue containing the major portion of the vitamin A and other primary alcohols and a small percentage of vitamin D and hydrocarbons. The petroleum ether fraction was extracted with fifteen 150 part portions of 95% methanol. After these extractions the petroleum ether fraction consisted chiefly of hydrocarbons and a relatively small amount of vitamin A and vitamin D. The combined 95% methanol fractions contained the major portion of the vitamin D present in the original unsaponifiable fraction.

- The vitamin D fraction after being freed of methanol was saponifiedby adding 9 parts of 5% methanolic KOH to 1 part of the traction and refluxing for about 20 minutes. The saponifled mass was cooled to room temperature and 18 parts of water added. The mass was then extracted three times with petroleum ether, and

the petroleum ether extracts combined and washed once without shaking with an equal volume of water. The solution was then washed once with thorough shaking with one-half the volume of 3% aqueous KOH, then three times with the same volume of water without shaking, and finally three times with water with shaking.

The washings were discarded. The residue which was obtained on removing the petroleum ether from the vitamin D fraction contained 1,200,000

"U. '8. P. units of vitamin D per gram. metal compound, such free carboxyl groups will The vitamin .A .i'raction was saponified and extracted as described in connection with the vitamin D fraction, thereby yielding a vitamin units of vitamin A/Em.

' Example 11' A fraction having a potency of 1,180,000 '0'. S. P.

of two hours. The resulting concentrates which i were obtained were much more potent than the original concentrate. The resulting vitamin D concentrate contained over 900,000 units of vita min D/gm. and the vitamin A concentrate contained 850,000 units vitamin A/g'm.

Example III of vitamin A/gm. was treated much the same as in Example I except that benzoic anhydride was used in place of phthalic anhydrlde as the acylat-' min D ester in the same. manner as disclosed with respect to the vitamin A esters. Moreover, the same type of solvents may be employed in removing the vitamin D esters from the hydrocarbons, such as squalene, as is used in extracting the unesterifled vitamin D.

The process of this invention may be used to prepare concentrates of vitamin A and concentrates of vitamin D from any material containing both vitamins A and D, It is particularly di rected to the treating of the unsaponifiable portion of fish liver oils and other marine oils, such as are obtained from'cod, halibut, herring, sardine, tuna, swordfish, shark, ling cod, jew fish,

blue whale, pollack, mackerel, etc.

The highly potent vitamin A and vitamin concentrates produced by the process of my invention may be used for the same purposes for which ordinary concentrates are adapted. They may be used for pharmaceutical purposes, for fortifying foods, or any other similar purpose. Varying amounts of the vitamin A concentrate and of the vitamin D concentrate may be admixed with each other to obtain products containing any ratio of vitamin A to vitamin D desired. Likewise the vitamin A concentrate may be admixed with synthetic forms of vitamin D, such as activated ergosterol, cholesterol, 'I-dehydro cholesterol, etc.

Although the unsaponifiable matter may be obtained from the oils in any usual way, it is preferred to use either the process disclosed and claimed in copending application of Buxton and Simons, Serial No. 333,114, filed May 3, 1940, now Patent No. 2,318,748, or the process disclosed and claimed in copending application of Buxton and Colman, Serial No. 350,166, filed August '2, 1940, now Patent No. 2,318,749.

The unsaponifiable fraction of certain fish and fish liver oils contains various primary alcohols other than vitamin A. For example, such fractions derived from cod liver oil contain batyl, chimyl and selachyl alcohols which will react with the acylating agents employed in the process of the invention; consequently these primary alcohols willbe esterified and extracted out with the vitamin. A. On saponification of the vitamin A ester and subsequent extraction of the alcoholic form of vitamin, certain of the alcohols and particularly batyl alcohol will remain in the residue and thus be separated from the vitamin A. v

The expressions units of vitamin A" and units of vitamin D" as used hereinabove refer to the U. S. P. units of the saidvitamins.

For the sake of brevitythe expression vitamin concentra is used in the appended claims to connote the unsaponifiable fraction of a fish or fish liver oil produced by saponification procedures. bon solvent" is employed in the appended claims The expression aliphatic hydrocarto connote straight chain, branched chain and/or alicyclic hydrocarbon solvents.

Since certain changes may be made in carrying out the above process without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.

It is also to be understood that the followin claims are intended to cover all the generic and specific features of the invention herein described, and all statements of the scope of the invention, which as a matter of language might be said to falltherebetween.

Having described my invention what I claim as new and desire to secure by Letters Patent is:

1. A process of treating, a vitamin A and D concentrate to separate vitamin A from vitamin D, which comprises esterifying vitamin A to the substantial exclusion of the vitamin D with an acylating agent selected from the group consisting of the anhydrides and acyl halides of aromatic acids and aliphatic dicarboxylic acids and extracting an aliphatic hydrocarbon solvent solution of the reactionmass with an acne-- ous solution of a water-miscible organic solvent to selectively remove esterified'vitamin A.

2. A process of treating a vitamin A and D concentrate to separate vitamin A from vitae .min D, which comprises esterifying vitamin A to the substantial exclusion of the vitamin D with an acylating agent .selected from the group consisting of the anhydrides and acyl halides of aromatic acids and aliphatic dicarboxylic acids and extracting an aliphatic hydrocarbon solvent solution of the reaction mass with a lower aliphatic alcohol containing more than 5% moisture to selectively remove esterifled vitamin A.

' 3. A process of treating a vitamin A and D concentrate to separate vitamin A from vitamin vitamin'wA to vitamin D with D, which comprises esterifying the substantial exclusion of the an acylatingageut selected from the group consisting of the anhydridesand acyl halides of aromatic acids, and aliphatic dicarboxylic acids,

extracting an aliphatic hydrocarbon solvent 50- lution of the reaction mass with a lower aliphatic alcohol containing more than 5% moisture to selectively remove esterified vitamin A, and removing vitamin D from the residual mass by extraction with a. lower aliphatic alcohol containing no more than 5% moisture.

4. A process of treating a vitamin A and'D concentrate to separate vitamin A from vitamin D, which comprises esterifying vitamin A to the substantial exclusion of the vitamin D, in the presence of pyridine, with an acylating agent selected from the group consisting of the anhydrides and acyl halides of aromatic acids and aliphatic dicarboxylic acids, extracting an aliphatic hydro.- carbon solvent solution of the reaction mass with a lower aliphatic alcohol containing more than 5%moisture to selectively remove esterified vitamin A, and removing vitamin D from the residual mass by extraction with a lower aliphatic alcohol containing no more than 5% moisture.

5. A process of treating a vitamin A and D concentrate to separate vitamin A from vitamin D, which comprises esterifying vitamin A to the substantial exclusion of the vitamin D with an acylating agent selected from the group consisting of the anhydrides and acyl halides of aromatic acids and aliphatic dicarboxylic acids, extracting an aliphatic hydrocarbon solvent solution of the reaction mass with 85% to 94% methanol to selectively remove esterified vitamin A, and removing vitamin D from the residual mass by extraction with a lower aliphatic alcohol containing no more than moisture.

6. A process of treating a vitamin A and D concentrate to separate vitamin A from vitamin D,

tively remove esterifled vitamin A, and removing vitamin D from the residual mass by extraction with a lower than 5% moisture. I

7. A process of treating a'vltamin A and D concentrate to separate vitamin A from vitamin D, which comprises esterifying vitamin A to the substantial exclusion of vitamin D with phthalic anhydride in the presence of pyridine, removing the pyridine, extracting an aliphatic hydrocarbon solution of the partially esterified mass with 85% to 94% methanol to remove the esterified vitamin A, and removing the vitamin D from the residual.

mass by extraction with 95% to 100% methanol.

8. A process of treating a vitamin A and D c0n-, centrate to separate vitamin A from vitamin D, which comprises steriiying vitamin A to the substantial exclusion of vitamin D with benzoyl chloride in the presence or pyridine, removing the pyridine, extracting an aliphatic hydrocarbon solution of the partially esteriiied mass with 85% to 94% methanol to remove the esteriiled vitamin aliphatic alcohol containing no more moving the vitamin D from the residual A, and removing the vitamin D from the residual mass by extraction with 95% to 100% methanol.

9. A process of treating a vitamin A and D con- I centrate to separate vitamin A from vitamin D. which comprises esterifying vitamin A to the substantial exclusion of vitamin D with phthalic anhydride in the presence of pyridine, removing the pyridine, extracting a heptane solution 01. the

partially esterifled mass with to 94% methanol to remove the esterifled vitamin A, and remass by extraction with 95% to methanol.

10. A process of treating a vitamin A and D concentrate to separate vitamin A from vitamin D, which comprises esterifying vitamin A to the substantial exclusion of vitamin D with phthalic anhydride in the presence of pyridine, removing the pyridine, extracting a cyclohexane solution of the partially esterified mass with 85% to 94% methanol to remove the esterified vitamin A, and removing the vitamin D from the residual mass by extraction with 95% to 100% methanol.

11. A process of treating a vitamin A and D concentrate to separate vitamin A from vitamin D, which comprises esterifying vitamin A to the substantial exclusion of vitamin D with an anhydride of a dicarboxylic acid in the presence of pyridine, removing the pyridine, forming an alkali metal salt with the carboxyl groups remaining free in the vitamin A esters, extracting an aliphatic hydrocarbon solution of the partially esterfled mass with methanol containing more than 5% moisture to remove the esteritled vitamin A, and removing vitamin D from the residual mass by extraction with methanol containing no more than5% moisture.

LORAN O. BUXTON. 

